Skip to main content
SpringerLink
Log in

Visualizing the Dynamics of Cell Division by Live Imaging Drosophila Larval Brain Squashes

  • Protocol
  • First Online:
Mitosis

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2415))

Abstract

The dramatic changes of subcellular structures during mitosis are best visualized by live imaging. In general, live imaging requires the expression of proteins of interest fused to fluorophores and a model system amenable to live microscopy. Drosophila melanogaster is an attractive model in which to perform live imaging because of the numerous transgenic stocks bearing fluorescently tagged transgenes as well as the ability to precisely manipulate gene expression. Traditionally, the early Drosophila embryo has been used for live fluorescent analysis of mitotic events such as spindle formation and chromosome segregation. More recent studies demonstrate that the Drosophila third instar neuroblasts have a number of properties that make them well suited for live analysis: (1) neuroblasts are distinct cells surrounded by plasma membranes; (2) neuroblasts undergo a complete cell cycle, consisting of G1, S, G2, and M phases; and (3) neuroblasts gene expression is not influenced by maternal load, and so the genetics are therefore relatively more simple. Finally, the Drosophila neuroblast is arguably the best system for live imaging asymmetric stem cell divisions. Here, we detail a method for live imaging Drosophila larval neuroblasts.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 109.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 139.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 199.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Rieder CL, Khodjakov A (2003) Mitosis through the microscope: advances in seeing inside live dividing cells. Science 300:91–96. https://doi.org/10.1126/science.1082177

    Article  CAS  PubMed  Google Scholar 

  2. Zirkle RE (1970) Ultraviolet-microbeam irradiation of newt-cell cytoplasm: spindle destruction, false anaphase, and delay of true anaphase. Radiat Res 41:516–537. https://doi.org/10.2307/3572841

    Article  CAS  PubMed  Google Scholar 

  3. Hayden JH, Bowser SS, Rider CL (1990) Kinetochores capture astral microtubules during chromosome attachment to the mitotic spindle: direct visualization in live newt lung cells. J Cell Biol 111:1039–1045. https://doi.org/10.1083/jcb.111.3.1039

    Article  CAS  PubMed  Google Scholar 

  4. Gönczy P, Schnabel H, Kaletta T, Amores AD, Hyman T et al (1999) Dissection of cell division processes in the one cell stage Caenorhabditis elegans embryo by mutational analysis. J Cell Biol 144:927–946. https://doi.org/10.1083/jcb.144.5.927

    Article  PubMed  PubMed Central  Google Scholar 

  5. Sullivan W, Minden JS, Alberts BM (1990) Daughterless-abo-like, a Drosophila maternal-effect mutation that exhibits abnormal centrosome separation during the late blastoderm divisions. Development 110:311–323

    Article  CAS  Google Scholar 

  6. Ellenberg J, Siggia ED, Moreira JE, Smith CL, Presley JF et al (1997) Nuclear membrane dynamics and reassembly in living cells: targeting of an inner nuclear membrane protein in interphase and mitosis. J Cell Biol 138:1193–1206. https://doi.org/10.1083/jcb.138.6.1193

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Straight AF, Sedat JW, Murray AW (1998) Time-lapse microscopy reveals unique roles for kinesins during anaphase in budding yeast. J Cell Biol 143:687–694. https://doi.org/10.1083/jcb.143.3.687

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Duffy JB (2002) GAL4 system in Drosophila: a fly geneticist’s Swiss army knife. Genesis 34:1–15. https://doi.org/10.1002/gene.10150

    Article  CAS  PubMed  Google Scholar 

  9. Fasulo B, Sullivan W (2014) Live confocal analysis of mutant- and drug-treated Drosophila embryos. Methods Mol Biol 1075:243–255. https://doi.org/10.1007/978-1-60761-847-8_12

    Article  PubMed  Google Scholar 

  10. Schmied C, Tomancak P (2016) Sample preparation and mounting of Drosophila embryos for multiview light sheet microscopy. Methods Mol Biol 1478:189–202. https://doi.org/10.1007/978-1-4939-6371-3_10

    Article  CAS  PubMed  Google Scholar 

  11. Yamashita YM, Jones DL, Fuller MT (2003) Orientation of asymmetric stem cell division by the APC tumor suppressor and centrosome. Science 301:1547–1550. https://doi.org/10.1126/science.1087795

    Article  CAS  PubMed  Google Scholar 

  12. Hughes SE, Hawley RS (2017) Live imaging of meiosis I in late-stage Drosophila melanogaster oocytes. Methods Mol Biol 1471:255–264. https://doi.org/10.1007/978-1-4939-6340-9_14

    Article  CAS  PubMed  Google Scholar 

  13. Martin JL, Sanders EN, Moreno-Roman P, Koyama LAJ, Balachandra S et al (2018) Long-term live imaging of the Drosophila adult midgut reveals real-time dynamics of division, differentiation and loss. elife. https://doi.org/10.7554/eLife.36248

  14. Restrepo S, Zartmann JJ, Basler K (2016) Cultivation and live imaging of Drosophila imaginal discs. Methods Mol Biol 1478:203–213. https://doi.org/10.1007/978-1-4939-6371-3_11

    Article  CAS  PubMed  Google Scholar 

  15. Moutinho-Pereira S, Matos I, Maiato H (2010) Drosophila S2 cells as a model system to investigate mitotic spindle dynamics, architecture, and function. Methods Cell Biol 97:243–257. https://doi.org/10.1016/S0091-679X(10)97014-3

    Article  CAS  PubMed  Google Scholar 

  16. Lerit DA, Plevock KM, Rusan NM (2014) Live imaging of Drosophila larval neuroblasts. J Vis Exp 89. https://doi.org/10.3791/51756

  17. Buffin E, Lefebvre C, Huang J, Gagou ME, Karess RE (2005) Recruitment of Mad2 to the kinetochore requires the Rod/Zw10 complex. Curr Biol 15:856–861. https://doi.org/10.1016/j.cub.2005.03.052

    Article  CAS  PubMed  Google Scholar 

  18. Royou A, Gagou ME, Karess R, Sullivan W (2010) BubR1- and polo-coated DNA tethers facilitate poleward segregation of acentric chromatids. Cell 140:235–245. https://doi.org/10.1016/j.cell.2009.12.043

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Oliveira RA, Kotadia S, Tavares A, Mirkovic M, Bowlin K et al (2014) Centromere-independent accumulation of cohesin at ectopic heterochromatin sites induces chromosome stretching during anaphase. PLoS Biol 12. https://doi.org/10.1371/journal.pbio.1001962

  20. Emre D, Tarracol R, Poncet A, Rahman Z, Karess RE (2011) A mitotic role for Mad1 beyond the spindle checkpoint. J Cell Sci 124:1664–1671. https://doi.org/10.1242/jcs.081216

    Article  CAS  PubMed  Google Scholar 

  21. Karg T, Elting MW, Vicars H, Dumont S, Sullivan W (2017) The chromokinesin Klp3a and microtubules facilitate acentric chromosome segregation. J Cell Biol 216:1597–1608. https://doi.org/10.1083/jcb.201604079

    Article  PubMed  PubMed Central  Google Scholar 

  22. Katsani KR, Karess RE, Dostatni N, Doye V (2008) In vivo dynamics of Drosophila nuclear envelope components. Mol Biol Cell 19:3652–3666. https://doi.org/10.1091/mbc.E07-11-1162

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Karg T, Warecki B, Sullivan W (2015) Aurora B-mediated localized delays in nuclear envelope formation facilitate inclusion of late-segregating chromosome fragments. Mol Biol Cell 26:2227–2241. https://doi.org/10.1091/mbc.E15-01-0026

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Warecki B, Sullivan W (2018) Micronuclei formation is prevented by aurora B-mediated exclusion of HP1a from late-segregating chromatin in Drosophila. Genetics 210:171–187. https://doi.org/10.1534/genetics.118.301031

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Kotadia S, Montembault E, Sullivan W, Royou A (2012) Cell elongation is an adaptive response for clearing long chromatid arms from the cleavage plane. J Cell Biol 199:745–753. https://doi.org/10.1083/jcb.201208041

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Montembault É, Claverie MC, Bouit L, Landmann C, Jenkins J et al (2017) Myosin efflux promotes cell elongation to coordinate chromosome segregation with cell cleavage. Nat Commun 8:326. https://doi.org/10.1038/s41467-017-0337-6

    Article  PubMed  PubMed Central  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Molecular, Cell, and Developmental Biology, University of California, Santa Cruz, Santa Cruz, CA, USA

    Brandt Warecki, Ian Bast & William Sullivan

Authors
  1. Brandt Warecki
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ian Bast
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. William Sullivan
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to William Sullivan .

Editor information

Editors and Affiliations

  1. The Hormel Institute, University of Minnesota, Austin, MN, USA

    Edward H. Hinchcliffe

Rights and permissions

Reprints and permissions

Copyright information

© 2022 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Warecki, B., Bast, I., Sullivan, W. (2022). Visualizing the Dynamics of Cell Division by Live Imaging Drosophila Larval Brain Squashes. In: Hinchcliffe, E.H. (eds) Mitosis. Methods in Molecular Biology, vol 2415. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1904-9_3

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-1904-9_3

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-1903-2

  • Online ISBN: 978-1-0716-1904-9

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation